1. Field of the Invention
The present invention relates to polynucleotides that induce or promote regeneration of plants.
2. Description of the Related Art
A bibliography follows at the end of the Detailed Description of the Invention. The listed references are all incorporated herein by reference.
Organogenesis and embryogenesis are two pathways leading to plant regeneration in a plant tissue culture. Traditionally, these two pathways are achieved through the manipulation of the contents and ratios of hormones in a plant-cell-culture medium as well as environmental conditions of a plant cell culture. Today, it is well known that these hormones and environmental cues activate specific proteins that play pivotal roles in the initiation of organogenesis or embryogenesis. This understanding opens a new door to novel methods for manipulation of plant regeneration through a direct control of gene expression. For example, over-expression of Arabidopsis ESR1 and ESR2, which are transcriptional factors belonging to AP2 family, induces shoot regeneration in a plant-cell-culture medium that does not contain any cytokinins (Banno et al., 2001; U.S. Pat. Nos. 6,441,276; 6,407,312). Likewise, genes involved in cytokinin production, such as ipt, can be over-expressed to substitute for a cytokinin in a culture medium (Ooms et al., 1983, Smigocki and Owens, 1988; Ebinuma et al., 1997). Similarly, genes involved in transduction of cytokinin signals, such as cytokinin activated histidine kinase (CKI1) and two-component transcription activators (ARR1 and ARR2), can promote shoot regeneration in Arabidopsis (Kakimoto, 1996; Sakai et al., 2001; Hwang and Sheen, 2001; Imamura et al., 2003).
Gene transformation is an important tool of molecular biology as well as crop improvement. Although currently many plants can be transformed using methods such as Agrobacterium-mediated T-DNA conjugation and particle gun bombardment, the efficiency of those methods varies, and there is a need to improve transformation efficiency of important crops, such as cotton, maize and soybean. Improved transformation efficiency is especially important to perform a large-scale analysis of gene functions in these crops. Improved transformation efficiency can be achieved by a higher rate of successful transformation, easier selection of successful transformants, shorter time for such a selection, or lesser use of antibiotics or herbicides for such a selection, and will generate economical, industrial or academic benefits.
Cotton is an economically important crop but is one of the most difficult plants to transform. It usually takes about 1.5 years to produce transgenic seeds. No cotton gene has so far been reported to improve plant regeneration or transformation although some Arabidopsis genes have been reported to improve transformation efficiency of root explants of Arabidopsis (Bann et al., 2001; U.S. Pat. Nos. 6,441,276; 6,407,312). Therefore, there is a need for such a cotton gene.
Although currently antibiotics or herbicide resistance markers are almost exclusively used for selection of plant transformants (Yoder and Goldsbrough, 1994), they generally have negative effects on proliferation and differentiation and sometimes retard differentiation of adventitious shoots during the transformation process (Ebinuma et al., 1997). Further, they pose environmental or health risks (Bryant and Leather, 1992; Gressel, 1992; Flavell et al., 1992). The availability of new selection markers also facilitates stacking of multiple transgenic traits. Consequently, there have been considerable efforts to develop alternative selection systems for plant transformants. U.S. Pat. Nos. 6,441,276 and 6,407,312 disclose methods of selecting transformants using ESR genes.
Arabidopsis Wuschel was first described as a mutant defective in shoot apical meristem initiation and maintenance (Endrizzi et al., 1996; Laux et al., 1996; Mayer et al., 1998). The phenotype was attributed to mutation of a single gene (Wus) that encodes a homeodomain (“HD”) protein, a probable transcriptional factor (Mayer et al., 1998). Interestingly, although Arabidopsis contains at least 14 genes that are predicted to encode a highly similar HD domain at the N-terminus and a highly divergent sequence at the C-terminus to Wus gene, none of them could substitute for Wus in the Wuschel mutant. This indicates that these genes have different functions. Recently it was demonstrated that the mRNAs of these genes have unique expression profiles (Haecker et al., 2004). Except for Wus and Prs, the functions of these genes have not yet been defined. Although over-expression of the Arabidopsis Wus induces shoot or somatic embryo formation in Arabidopsis and rice (Zuo et al., 2002; Kamiya et al., 2003), not every HD domain protein has this property. The Arabidopsis PRS, for example, was required for flower development and its over-expression induces cell proliferation rather than shoot regeneration (Matsumoto and Okada, 2001). Another HD domain protein, At1g46480, was not able to induce shoot regeneration in a cytokinin-free medium (Table 1). A PCT publication WO 01/23575 A2 discloses several putative Wuschel homologues from maize and soybean. It was also shown that over-expression of Wuschel also induced shoot regeneration both in Arabidopsis and rice (Zuo et al., 2002; Kamiya et al., 2003).
Transcription factors generally consist of at least two modules that are often exchangeable between different members or classes. This type of chimeric transcriptional factors has been well documented in the literature. For example, VXE transcriptional factor is a fusion protein of a viral activation domain VP16, an E. coli LexA DNA binding domain and a human estrogen receptor regulatory domain (Zou and Chua, 2000). Surprisingly, a fusion protein containing the HD domain of GhCIR1 and the VP16 activation domain did not promote shoot regeneration (Table 5). This indicates that the HD domain alone is not sufficient for the shoot regeneration enhancing function of certain HD domain proteins. Phylogenic analysis showed that none of the three polynucleotides described in this disclosure, i.e., SEQ ID NOs: 1, 3 and 5, are closely related to Wuschel (FIG. 3).
Development of efficient and simple transformation techniques has made great contribution to rapid advance in molecular genetics in Arabidopsis. Currently, both in planta (flora dip) and root explant methods allow a large number of genes to be mutated or transformed (Clough and Bent, 1998; Valvekens et al., 1988). For example, Banno and Chua (2001) have identified an Arabidopsis gene by functional screening of an Arabidopsis cDNA library using Arabidopsis root explants. On the other hand, we identified a non-Arabidopsis (GhCIR1) gene by direct functional screening using Arabidopsis root explants (Example 2).